Method for Using Aqueous Two-Phase System for the Isolation, Purification and/or Concentration of Short Nucleic Acid Fragments

Abstract

The present invention relates to methods of using aqueous two-phase system (ATPS) for the isolation, concentration and/or purification of short nucleic acid fragment having about or less than 250 base pairs (bp). In one embodiment, the present invention provides a composition and kit for the purification of short nucleic acid fragments having about or less than 250 base pairs from nucleic acid-containing biological materials. In another embodiment, the present invention provides uses of certain salts and/or polymers in a two-phase system for the purification of short nucleic acid fragments having about or less than 250 base pairs from nucleic acid containing biological materials.

1 . A composition for isolating and concentrating nucleic acid fragments of 250 bp or less from a sample, comprising a first phase component and a second phase component capable of forming an aqueous two phase system (ATPS) when the components are dissolved in an aqueous solution, wherein the first phase component is a polymer dissolving in a first phase at a concentration of 5-20 wt %, and the second phase component is a salt dissolving in a second phase at a concentration of 5-35 wt %, wherein when said nucleic acid fragments are mixed with said ATPS, said fragments are concentrated in one of the two phases. 
     
     
         2 . The composition of  claim 1 , wherein said polymer is selected from the group consisting of polyalkylene glycols, poly (oxy alky lene)polymers, poly(oxyalkylene)copolymers, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methylether, alkoxylated surfactants, alkoxylated starches, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicone-modified poly ethers, poly N-isopropylacrylamide, polyethylene glycol, polypropylene glycol, polyacrylate, dextran, UCON™ polymer and Ficoll™ polymer. 
     
     
         3 . The composition of  claim 1 , wherein said salt is selected from the group consisting of kosmotropic salts, chaotropic salts, inorganic salts having a cation of trimethyl ammonium, triethyl ammonium, tripropyl ammonium, tributyl ammonium, tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium or tetrabutyl ammonium, and an anion of phosphate, sulphate, nitrate, chloride or hydrogen carbonate, sodium chloride, sodium phosphate, potassium phosphate, sodium sulphate, potassium citrate, ammonium sulfate, sodium citrate, sodium acetate and ammonium acetate. 
     
     
         4 . The composition of  claim 1 , wherein said polymer is polyethylene glycol having a molecular weight ranges from 100 to 10,000 Da. 
     
     
         5 . The composition of  claim 1 , wherein said salt is one or more of potassium phosphate dibasic, potassium phosphate monobasic, sodium chloride, sodium sulphate, and sodium phosphate. 
     
     
         6 . The composition  claim 1 , wherein when the sample is mixed with the composition, the resulting mixture undergoes phase separation and nucleic acid fragments of 250 bp or less are concentrated in one of the two phases. 
     
     
         7 . The composition of  claim 6 , wherein nucleic acid fragments larger than 250 bp and fragments of 250 bp or less are concentrated in different phases of the ATPS. 
     
     
         8 . The composition of  claim 1 , wherein said nucleic acid fragments are selected from the group consisting of coding DNA, non-coding DNA, messenger RNA, ribosomal RNA, micro-RNA and transfer RNA. 
     
     
         9 . The composition of  claim 1 , wherein said nucleic acid fragments are cell-free DNA or circulating tumor DNA. 
     
     
         10 . The composition of  claim 1 , wherein the sample is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, urine, saliva, fecal matter, tear, sputum, nasopharyngeal mucus, vaginal discharge and penile discharge. 
     
     
         11 . A method for isolating and concentrating nucleic acid fragments of 250 bp or less from a sample, comprising:
 a) preparing the composition of  claim 1 ;   b) mixing the composition from step a) with an aqueous solution containing said sample, allowing the resulting mixture to partition into two phases, wherein said nucleic acid fragments are concentrated in one of the two phases; and   c) isolating said nucleic acid fragments from one of the two phases.   
     
     
         12 . A method for isolating and concentrating nucleic acid fragments of 250 bp or less from a sample, comprising:
 a) preparing a composition comprising a first phase component and a second phase component capable of forming an aqueous two phase system (ATPS) when the components are dissolved in an aqueous solution, wherein the first phase component is a polymer dissolving in a first phase at a concentration of 5-20 wt %, and the second phase component is a salt dissolving in a second phase at a concentration of 15-25 wt %;   b) mixing the composition from step a) with an aqueous solution containing said sample, allowing the resulting mixture to partition into two phases, wherein said nucleic acid fragments are concentrated in one of the two phases; and   c) isolating one of the two phases that is concentrated with said nucleic acid fragments.   
     
     
         13 . The method of  claim 12 , further comprising recovering the nucleic acid fragments from the phase in step c). 
     
     
         14 . The method of  claim 12 , wherein said polymer is selected from the group consisting of polyalkylene glycols, poly (oxy alky lene)polymers, poly(oxyalkylene)copolymers, polyvinyl pyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methylether, alkoxylated surfactants, alkoxylated starches, alkoxylated cellulose, alkyl hydroxyalkyl cellulose, silicone-modified poly ethers, poly N-isopropylacrylamide, polyethylene glycol, polypropylene glycol, polyacrylate dextran, UCON™ polymer and Ficoll™ polymer. 
     
     
         15 . The method of  claim 12 , wherein said salt is selected from the group consisting of kosmotropic salts, chaotropic salts, inorganic salts having a cation of trimethyl ammonium, triethyl ammonium, tripropyl ammonium, tributyl ammonium, tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium or tetrabutyl ammonium, and an anion of phosphate, sulphate, nitrate, chloride or hydrogen carbonate, sodium chloride, sodium phosphate, potassium phosphate, sodium sulphate, potassium citrate, ammonium sulfate, sodium citrate, sodium acetate and ammonium acetate. 
     
     
         16 . The method of  claim 12 , wherein said polymer is polyethylene glycol having a molecular weight ranges from 100 to 10,000 Da. 
     
     
         17 . The method of  claim 12 , wherein said salt is one or more of potassium phosphate dibasic, potassium phosphate monobasic, sodium chloride, sodium sulphate, and sodium phosphate. 
     
     
         18 . The method of  claim 12 , wherein in step b), nucleic acid fragments larger than 250 bp and fragments of 250 bp or less are concentrated in different phases of the ATPS. 
     
     
         19 . The method of  claim 12 , wherein said nucleic acid fragments are selected from the group consisting of coding DNA, non-coding DNA, messenger RNA, ribosomal RNA, micro-RNA and transfer RNA. 
     
     
         20 . The method of  claim 12 , wherein said nucleic acid fragments are cell-free DNA or circulating tumor DNA. 
     
     
         21 . The method of  claim 12 , wherein the sample is selected from the group consisting of blood, plasma, serum, cerebrospinal fluid, urine, saliva, fecal matter, tear, sputum, nasopharyngeal mucus, vaginal discharge and penile discharge. 
     
     
         22 . The method of  claim 12 , wherein said nucleic acid fragments are concentrated by at least 10 folds as compared to said sample.